羟乙基呱嗪乙硫磺酸对人骨髓间充质干细胞纯化培养的影响  

Effect of Hepes on purification and cultivation of human mesenchymal stem cells

作　　者：李敏霞[1] 李相中[2] 刘清芳[1] 

机构地区：[1]安阳市人民医院检验科,安阳455000 [2]安阳市中医药学校基础部,安阳455000

出　　处：《郑州大学学报（医学版）》2005年第3期477-479,共3页Journal of Zhengzhou University(Medical Sciences)

摘　　要：目的:探讨羟乙基呱嗪乙硫磺酸对人骨髓间充质干细胞(hMSC)纯化培养的影响。方法:采用全髓直接接种法分离培养13～18周胎龄水囊引产新鲜胎儿股骨骨髓,贴壁细胞达90%以上融合时消化传代。传代细胞分别接种于含100ml/L胎牛血清的LDMEM培养基(常规培养基)和加入15mmol/L羟乙基呱嗪乙硫磺酸(Hepes)的常规培养基(改良培养基)中,观察细胞生长情况,用酸度计监测培养基pH值变化情况,流式细胞仪鉴定细胞纯度及CD抗原表达情况。并诱导改良培养基培养的hMSC向成骨细胞分化,酶细胞化学法和全自动生化仪检测碱性磷酸酶(ALP)活性,鉴定hMSC的成骨分化能力。结果:2种培养基培养的hMSC在细胞形态、生长特性、CD抗原表达等方面相似,但改良培养基由于pH值较恒定而能提高hMSC纯度及细胞增殖速度,且诱骨分化后可形成矿化结节,ALP染色阳性。结论:Hepes能提高全髓直接接种法培养的hMSC纯度及细胞增殖速度,简化骨髓细胞分离程序,减少细胞损伤及受污染机会,利于hMSC培养的推广应用。Aim: To explore the effect of Hepes on the purification and cultivation of human mesenchymal stem cells(hMSC).Methods: By using holo-marrow direct inoculation, the fresh femoral bone marrow of 13～18 weeks age embryo was cultured. When the adherence cells were confluenced to 90%, the cells were digested and subcultured. The passage cells were inoculated in conventional L-DMEM media and in improved media that was L-DMEM pretreated with Hepes at 15 mmol/L, respectively. The growth of cells was observed. The pH value was measured. The purity of cells was analyzed by using flow cytometry. The cultural MSC were induced to differentiate into osteoblast.By using enzyme cytochemistry and automatic biochemistry analyzer, the alkaline phosphatase activities were detected, and the osteogenesis differentiation capability of MSC was determined.Results: hMSC cultured in 2 mediums had resembling cell configuration,growth characteristics and surface antigen presentation. But in the improvement media, the purity coefficient and multiplication speed of MSC were elevated. Conclusion: Hepes can increase the purity coefficient and multiplication speed of hMSC, reduce the separating procedures of bone marrow cells, and decrease cell damage.

关 键 词：间充质干细胞 体外培养 人骨髓 羟乙基呱嗪乙硫磺酸 

分 类 号：R329.4[医药卫生—人体解剖和组织胚胎学]